Prolonged Heat Stress during Winter Diapause Alters the Expression of Stress-Response Genes in Ostrinia nubilalis (Hbn.).

During diapause, a state of temporarily arrested development, insects require low winter temperatures to suppress their metabolism, conserve energy stores and acquire cold hardiness. A warmer winter could, thus, reduce diapause incidence and duration in many species, prematurely deplete their energy reserves and compromise post-diapause fitness. In this study, we investigated the combined effects of thermal stress and the diapause program on the expression of selected genes involved in antioxidant defense and heat shock response in the European corn borer Ostrinia nubilalis. By using qRT-PCR, it has been shown that response to chronic heat stress is characterized by raised mRNA levels of grx and trx, two important genes of the antioxidant defense system, as well as of hsp70 and, somewhat, of hsp90, two major heat shock response proteins. On the other hand, the expression of hsc70, hsp20.4 and hsp20.1 was discontinuous in the latter part of diapause, or was strongly controlled by the diapause program and refractory to heat stress, as was the case for mtn and fer, genes encoding two metal storage proteins crucial for metal ion homeostasis. This is the first time that the effects of high winter temperatures have been assessed on cold-hardy diapausing larvae and pupae of this important corn pest.

Acclimations to Cold and Warm Conditions Differently Affect the Energy Metabolism of Diapausing Larvae of the European Corn Borer Ostrinia nubilalis (Hbn.).

The European corn borer Ostrinia nubilalis is a pest species, whose fifth instar larvae gradually develop cold hardiness during diapause. The physiological changes underlying diapause progression and cold hardiness development are still insufficiently understood in insects. Here, we follow a complex of changes related to energy metabolism during cold acclimation (5°C) of diapausing larvae and compare this to warm-acclimated (22°C) and non-diapause controls. Capillary electrophoresis of nucleotides and coenzymes has shown that in gradually cold-acclimated groups concentrations of ATP/ADP and, consequently, energy charge slowly decrease during diapause, while the concentration of AMP increases, especially in the first months of diapause. Also, the activity of cytochrome c oxidase (COX), as well as the concentrations of NAD+ and GMP, decline in cold-acclimated groups, until the latter part of diapause, when they recover. Relative expression of NADH dehydrogenase (nd1), coenzyme Q-cytochrome c reductase (uqcr), COX, ATP synthase (atp), ADP/ATP translocase (ant), and prohibitin 2 (phb2) is supressed in cold-acclimated larvae during the first months of diapause and gradually increases toward the termination of diapause. Contrary to this, NADP+ and UMP levels significantly increased in the first few months of diapause, after gradual cold acclimation, which is in connection with the biosynthesis of cryoprotective molecules, as well as regeneration of small antioxidants. Our findings evidence the existence of a cold-induced energy-saving program that facilitates long-term maintenance of larval diapause, as well as gradual development of cold hardiness. In contrast, warm acclimation induced faster depletion of ATP, ADP, UMP, NAD+, and NADP+, as well as higher activity of COX and generally higher expression of all energy-related genes in comparison to cold-acclimated larvae. Moreover, such unusually high metabolic activity, driven by high temperatures, lead to premature mortality in the warm-acclimated group after 2 months of diapause. Thus, our findings strongly support the importance of low temperature exposure in early diapause for gradual cold hardiness acquisition, successful maintenance of the resting state and return to active development. Moreover, they demonstrate potentially adverse effects of global climate changes and subsequent increase in winter temperatures on cold-adapted terrestrial organisms in temperate and subpolar regions.

Effect of Cold Acclimation on Selected Metabolic Enzymes During Diapause in The European Corn Borer Ostrinia nubilalis (Hbn.).

The European corn borer, Ostrinia nubilalis Hbn., is a pest Lepidopteran species whose larvae overwinter by entering diapause, gradually becoming cold-hardy. To investigate metabolic changes during cold hardening, activities of four metabolic enzymes - citrate synthase (CS), lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in whole-body homogenates of pupae, non-diapausing and diapausing larvae acclimated to 5 °C, -3 °C and -16 °C. The highest CS activity was detected in non-diapausing larvae, reflecting active development, while the highest in vitro LDH activity was recorded in diapausing larvae at temperatures close to 0 °C, evidencing a metabolic switch towards anaerobic metabolism. However, in-gel LDH activity showed that production of pyruvate from lactate is triggered by sub-zero temperatures. The activities of both aminotransferases were highest in non-diapausing larvae. Our findings suggest that during diapause and cold hardening the aminotransferases catalyse production of L-alanine, an important cryoprotectant, and L-aspartate, which is closely tied to both transamination reactions and Krebs cycle. The results of this study indicate that, during diapause, the activity of metabolic enzymes is synchronized with exogenous factors, such as temperatures close to 0 °C. These findings support the notion that diapause is metabolically plastic and vibrant, rather than simply a passive, resting state.